Methods and compositions for oral administration of proteins

ABSTRACT

This invention provides compositions that include a protein and at least two protease inhibitors, method for treating diabetes mellitus, and methods for administering same, and methods for oral administration of a protein with an enzymatic activity, including orally administering same.

FIELD OF INVENTION

This invention provides oral compositions comprising a protein and atleast two protease inhibitors and a method for administering same.

BACKGROUND OF THE INVENTION

Due to improved biotechnology, the accessibility of biologically activepeptides to the pharmaceutical industry has increased considerably.However, a limiting factor in the development of peptide drugs is therelative ineffectiveness when given perorally. Almost all peptide drugsare parenterally administered, although parenterally administeredpeptide drugs are often connected with low patient compliance.

Insulin is a medicament used to treat patients suffering from diabetes,and is the only treatment for insulin-dependent diabetes mellitus.Diabetes Mellitus is characterized by a pathological condition ofabsolute or relative insulin deficiency, leading to hyperglycemia, andis one of the main threats to human health in the 21st century. Theglobal figure of people with diabetes is set to rise to 220 million in2010, and 300 million in 2025. Type I diabetes is caused primarily bythe failure of the pancreas to produce insulin. Type II diabetes,involves a lack of responsiveness, of the body to the action of insulin.

Approximately 20%-30% of all diabetics use daily insulin injections tomaintain their glucose levels. An estimated 10% of all diabetics aretotally dependent on insulin injections.

Currently, the only route of insulin administration is injection. Dailyinjection of insulin is causes considerable suffering for patients. Sideeffects such as lipodystrophy at the site of the injection, lipatrophy,lipohypertrophy, and occasional hypoglycemia are known to occur. Inaddition, subcutaneous administration of insulin does not typicallyprovide the fine continuous regulation of metabolism that occursnormally with insulin secreted from the pancreas directly into the livervia the portal vein.

The present invention addresses the need for an alternate solution foradministration of insulin.

SUMMARY OF THE INVENTION

This invention provides, in one embodiment, a composition comprising aprotein or a combination of proteins having a molecular weight of up to100,000 Daltons and a first protease inhibitor and a second proteaseinhibitor.

In another embodiment, the present invention provides a method for oraladministration of a protein having a molecular weight up to 100,000Daltons to a subject, whereby a substantial fraction of the proteinretains its activity after absorption, through an intestinal mucosalbarrier of a subject, comprising administering orally to a subject apharmaceutical composition comprising the protein and a first proteaseinhibitor and a second protease inhibitor.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a subject, comprising administering orallyto a subject a pharmaceutical composition comprising insulin, Exenatide,or a combination thereof and a first protease inhibitor and a secondprotease inhibitor, thereby treating diabetes mellitus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-I) is a set of bar graphs showing the changes in blood glucoselevels (FIG. 1 A-C), blood insulin levels (FIG. 1 D-F) and bloodC-peptide levels (FIG. 1 G-I) after treatment with the formulations ofthe invention.

In FIG. 1 A-C blood glucose levels were significantly reduced in humansubjects treated with formulation (3) (8 mg insulin, 150 mg EDTA, 150000KIU Aprotinin, 125 mg SBTI in 1 ml fish oil in a soft-gel capsule(SwissCup).

FIG. 1 D-F shows that total blood insulin was significantly higherespecially between 220-300 minutes in human subjects treated withformulation (3).

FIG. 1 G-I, shows that blood C-peptide levels were significantly reducedin human subjects treated with formulation (3).

DETAILED DESCRIPTION OF THE INVENTION

This invention provides compositions and methods comprising a proteinand at least two protease inhibitors. In another, embodiment, thepresent invention provides compositions and methods comprising a proteinand a first protease inhibitor and a second protease inhibitor. Inanother, embodiment, the present invention provides compositions andmethods comprising a protein having a molecular weight of up to 100,000Daltons and a first protease inhibitor and a second protease inhibitor.

In another, embodiment, the protein of the present invention has amolecular weight of 1,000-5,000 Daltons. In another, embodiment, theprotein of the present invention has a molecular weight of 5,000-10,000Daltons. In another, embodiment, the protein of the present inventionhas a molecular weight of 10,000-20,000 Daltons. In another, embodiment,the protein of the present invention has a molecular weight of20,000-30,000 Daltons. In another, embodiment, the protein of thepresent invention has a molecular weight of 40,000-50,000 Daltons. Inanother, embodiment, the protein of the present invention has amolecular weight of 50,000-60,000 Daltons. In another, embodiment, theprotein of the present invention has a molecular weight of 60,000-70,000Daltons. Ia another, embodiment, the protein of the present inventionhas a molecular weight of 70,000-80,000 Daltons. In another, embodiment,the protein of the present invention has a molecular weight of80,000-90,000 Daltons. In another, embodiment, the protein of thepresent invention has a molecular weight of 90,000-100,000 Daltons. Inanother, embodiment, the protein of the present invention has amolecular weight of 100,000-150,000 Daltons.

In another embodiment, the protein has a molecular weight (MW) of 1-50kilodalton (kDa). In another embodiment, the MW is 1-45 kDa. In anotherembodiment, the MW is 1-40 kDa. In another embodiment, the MW is 1-35kDa. In another embodiment, the MW is 1-30 kDa. In another embodiment,the MW is 1-25 kDa. In another embodiment, the MW is 1-20 kDa. Inanother embodiment, the MW is 10-50 kDa. In another embodiment, the MWis 15-50 kDa. In another embodiment, the MW is 20-50 kDa. In anotherembodiment, the MW is 25-50 kDa. In another embodiment, the MW is 30-50kDa. In another embodiment, the MW is 35-50 kDa. In another embodiment,the MW is 1-100 kDa. In another embodiment, the MW is 1-90 kDa. Inanother embodiment, the MW is 1-80 kDa. In another embodiment, the MW is1-70 kDa. In another embodiment, the MW is 1-60 kDa. In anotherembodiment, the MW is 10-100 kDa. In another embodiment, the MW is15-100 kDa. In another embodiment, the MW is 20-100 kDa. In anotherembodiment, the MW is 25-100 kDa. In another embodiment, the MW is30-100 kDa. In another embodiment, the MW is 10-80 kDa. In anotherembodiment, the MW is 15-80 kDa. In another embodiment, the MW is 20-80kDa. In another embodiment, the MW is 25-80 kDa. In another embodiment,the MW is 30-80 kDa. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the MW is less than 20 kDa. In anotherembodiment, the MW is less than 25 kDa. In another embodiment, the MW isless than 30 kDa. In another embodiment, the MW is less than 35 kDa. Inanother embodiment, the MW is less than 40 kDa. In another embodiment,the MW is less than 45 kDa. In another embodiment, the MW is less than50 kDa. In another embodiment, the MW is less than 55 kDa. In anotherembodiment, the MW is less than 60 kDa. In another embodiment, the MW isless than 65 kDa. In another embodiment, the MW is less than 70 kDa. Inanother embodiment, the MW is less than 75 kDa. In another embodiment,the MW is less than 80 kDa. In another embodiment, the MW is less than85 kDa. In another embodiment, the MW is less than 90 kDa. In anotherembodiment, the MW is less than 95 kDa. In another embodiment, the MW isless than 100 kDa.

In another, embodiment, the protein of the present invention is insulin.In one embodiment, the insulin of methods and compositions of thepresent invention is human insulin. In another embodiment, the insulinis recombinant insulin. In another embodiment, the insulin isrecombinant human insulin. In another embodiment, the insulin is bovineinsulin. In another embodiment, the insulin is porcine insulin. Inanother embodiment, the insulin is whale insulin. In another embodiment,the insulin is a metal complex of insulin (e.g. a zinc complex ofinsulin, protamine zinc insulin, or globin zinc).

In another embodiment, the insulin is regular insulin. In anotherembodiment, the insulin is fast-acting insulin. In another embodiment,the insulin is lente insulin. In another embodiment, the insulin issemilente insulin. In another embodiment, the insulin is Ultralenteinsulin. In another embodiment, the insulin is NPH insulin. In anotherembodiment, the insulin is glargine insulin. In another embodiment, theinsulin is lispro insulin. In another embodiment, the insulin is aspartinsulin. In another embodiment, the insulin is a combination of two ormore of any of the above types of insulin. In another embodiment, theinsulin is any other type of insulin known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, the amount of insulin utilized in methods andcompositions of the present invention is 0.5-3 units (u)/kg in humans.In one embodiment, the units used to measure insulin in methods andcompositions of the present invention are USP Insulin Units. In oneembodiment, the units used to measure insulin are milligrams. In anotherembodiment, one international Unit (IU) of Insulin is equivalent to 45.5mg insulin.

In another embodiment, the amount of insulin is 0.1-1 u/kg. In anotherembodiment, the amount is 0.2-1 u/kg. In another embodiment, the amountis 0.3-1 u/kg. In another embodiment, the amount is 0.5-1 u/kg. Inanother embodiment, the amount is 0.1-2 u/kg. In another embodiment, theamount is 0.2-2 u/kg. In another embodiment, the amount is 0.3-2 u/kg.In another embodiment, the amount is 0.5-2 u/kg. In another embodiment,the amount is 0.7-2 u/kg. In another embodiment, the amount is 1-2 u/kg.In another embodiment, the amount is 1.2-2 u/kg. In another embodiment,the amount is 1-1.2 u/kg. In another embodiment, the amount is 1-1.5u/kg. In another embodiment, the amount is 1-2.5 u/kg. In anotherembodiment, the amount is 1-3 u/kg. In another embodiment, the amount is2-3 u/kg. In another embodiment, the amount is 1-5 u/kg. In anotherembodiment, the amount is 2-5 u/kg. In another embodiment, the amount is3-5 u/kg.

In another embodiment, the amount of insulin is 0.1 u/kg. In anotherembodiment, the amount is 0.2 u/kg. In another embodiment, the amount is0.3 u/kg. In another embodiment, the amount is 0.4 u/kg. In anotherembodiment, the amount is 0.5 u/kg. In another embodiment, the amount is0.6 u/kg. In another embodiment, the amount is 0.8 u/kg. In anotherembodiment, the amount is 1 u/kg. In another embodiment, the amount is1.2 u/kg. In another embodiment, the amount is 1.4 u/kg. In anotherembodiment, the amount is 1.6 u/kg. In another embodiment, the amount is1.8 u/kg. In another embodiment, the amount is 2 u/kg. In anotherembodiment, the amount is 2.2 u/kg. In another embodiment, the amount is2.5 u/kg. In another embodiment, the amount is 3 u/kg.

In another embodiment, the amount of insulin is 1-10 u. In anotherembodiment, the amount is 2-10 u. In another embodiment, the amount is3-10 u. In another embodiment, the amount is 5-10 u. In anotherembodiment, the amount is 1-20 u. In another embodiment, the amount is2-20 u. In another embodiment, the amount is 3-20 u. In anotherembodiment, the amount is 5-20 u. In another embodiment, the amount is7-20 u. In another embodiment, the amount is 10-20 u. In anotherembodiment, the amount is 12-20 u. In another embodiment, the amount is10-12 u. In another embodiment, the amount is 10-15 u. In anotherembodiment, the amount is 10-25 u. In another embodiment, the amount is10-30 u. In another embodiment, the amount is 20-30 u. In anotherembodiment, the amount is 10-50 u. In another embodiment, the amount is20-50 u. In another embodiment, the amount is 30-50 u. In anotherembodiment, the amount is 20-100 u. In another embodiment, the amount is30-100 u. In another embodiment, the amount is 100-150 u. In anotherembodiment, the amount is 100-250 u. In another embodiment, the amountis 100-300 u. In another embodiment, the amount is 200-300 u. In anotherembodiment, the amount is 100-500 u. In another embodiment, the amountis 200-500 u. In another embodiment, the amount is 300-500 u. In anotherembodiment, the amount is 200-1000 u. In another embodiment, the amountis 300-1000 u.

In another embodiment, the amount of insulin is 1 u. In anotherembodiment, the amount is 2 u. In another embodiment, the amount is 3 u.In another embodiment, the amount is 4 u. In another embodiment, theamount is 5 u. In another embodiment, the amount is 6 u. In anotherembodiment, the amount is 8 u. In another embodiment, the amount is 10u. In another embodiment, the amount is 12 u. In another embodiment, theamount is 14 u. In another embodiment, the amount is 16 u. In anotherembodiment, the amount is 18 u. In another embodiment, the amount is 20u. In another embodiment, the amount is 22 u. In another embodiment, theamount is 25 u. In another embodiment, the amount is 30 u. In anotherembodiment, the amount is 50 u. In another embodiment, the amount is 80u. In another embodiment, the amount is 100 u. In another embodiment,the amount is 120 u. In another embodiment, the amount is 140 u. Inanother embodiment, the amount is 160 u. In another embodiment, theamount is 180 u. In another embodiment, the amount is 200 u. In anotherembodiment, the amount is 300 u. In another embodiment, the amount is500 u.

In another embodiment, the protein is Exenatide. In another embodiment,the oral formulations of the present invention protect Exenatidebreakdown in the stomach. In another embodiment, Exenatide formulationof the invention controls blood sugar levels. In another embodiment,Exenatide formulation of the invention helps control blood sugar levels.In another embodiment, Exenatide formulation of the invention inducespancreatic production of insulin. In another embodiment, Exenatideformulation of the invention is used to treat type 2 (non-insulindependent) diabetes. In another embodiment, Exenatide formulation of theinvention is used in conjunction with other diabetes medicines.

In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 10 mcg tot mg. In another embodiment, the amount ofExenatide in a formulation as described herein is 10 mcg to 25 mcg. Inanother embodiment, the amount of Exenatide in a formulation asdescribed herein is 25 mcg to 50 mcg. In another embodiment, the amountof Exenatide in a formulation as described herein is 50 mcg to 60 mcg.In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 60 mcg to 70 mcg. In another embodiment, the amountof Exenatide in a formulation as described herein is 70 mcg to 80 mcg.In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 80 meg to 90 mcg. In another embodiment, the amountof Exenatide in a formulation as described herein is 90 mcg to 100 mcg.In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 100 mcg to 110 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 110 mcg to125 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 125 mcg to 150 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 150 mcg to175 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 175 mcg to 200 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 200 mcg to220 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 220 mcg to 240 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 240 mcg to260 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 260 mcg to 300 mcg.

In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 300 mcg to 350 meg. In another embodiment, theamount of Exenatide in a formulation as described herein is 350 mcg to400 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 400 mcg to 450 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 450 meg to500 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 550 mcg to 600 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 600 mcg to700 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 700 mcg to 800 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 800 mcg to900 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 900 meg to 1 mg.

In another embodiment, the Exenatide formulation as described herein istaken once a day. In another embodiment, the Exenatide formulation asdescribed herein is taken twice a day. In another embodiment, theExenatide formulation as described herein is taken three times a day. Inanother embodiment, the Exenatide formulation as described herein istaken four times a day. In another embodiment, the Exenatide formulationas described herein is taken five times a day. In another embodiment,one of skill in the art determines the dosage of a Exenatide formulationas described herein. In another embodiment, one of skill in the artdetermines the daily dose of a Exenatide formulation as describedherein. In another embodiment, one of skill in the art determines thedaily dosing regimen of a EXenatide formulation as described herein.

In another embodiment, the Exenatide formulation as described herein istaken at least 15 minutes before a meal. In another embodiment, theExenatide formulation as described herein is taken at least 30 minutesbefore a meal. In another embodiment, the Exenatide formulation asdescribed herein is taken at least 45 minutes before a meal. In anotherembodiment, the Exenatide formulation as described herein is taken atleast 60 minutes before a meal. In another embodiment, the Exenatideformulation as described herein is taken at least 75 minutes before ameal. In another embodiment, the Exenatide formulation as describedherein is taken at least 90 minutes before a meal. In anotherembodiment, the Exenatide formulation as described herein is taken atleast 100 minutes before a meal. In another embodiment, the Exenatideformulation as described herein is taken at least 120 minutes before ameal. In another embodiment, the Exenatide formulation as describedherein is taken at least 150 minutes before a meal. In anotherembodiment, the Exenatide formulation as described herein is taken atleast 180 minutes before a meal.

In another embodiment, the Exenatide formulation as described hereinreduces the side effects associated with an injectable dosage formcomprising Exenatide. In another embodiment, the Exenatide formulationas described herein reduces nausea as a side effect which is associatedwith an injectable dosage form comprising Exenatide. In anotherembodiment, the Exenatide formulation as described herein does notinduce nausea as a side effect which is associated with an injectabledosage form comprising Exenatide.

As provided herein, protease inhibitors protect the protein of thepresent invention from cleavage. In another embodiment, the presentinvention provides that protease inhibitors protect insulin of thepresent invention from cleavage. In another, embodiment, the presentinvention provides that protease inhibitors facilitate the proteinabsorption in the intestine of a subject. In another, embodiment, thepresent invention provides that protease inhibitors facilitate theabsorption of insulin in the intestine of a subject.

In another embodiment, the present invention provides the use of morethan two protease inhibitors in a single composition or a method. Inanother, embodiment, the present invention provides that the first andthe second protease inhibitors are serpins. In another, embodiment, thepresent invention provides that serpins are trypsin inhibitors. Inanother, embodiment, the present invention provides that the first andthe second protease inhibitors are serpins such as but not limited to:Alpha 1-antitrypsin, Antitrypsin-related protein, Alpha1-antichymotrypsin, Kallistatin, Protein C inhibitor, Cortisol bindingglobulin, Thyroxine-binding globulin, Angiotensinogen, Centerin, ProteinZ-related protease inhibitor, Vaspin, Monocyte neutrophil elastaseinhibitor, Plasminogen activator inhibitor-2, Squamous cell carcinomaantigen-1 (SCCA-1), Squamous cell carcinoma antigen-2 (SCCA-2), Maspin,PI-6, Megsin, PI-8, PI-9, Bomapin, Yukopin, Hurpin/Headpin,Antithrombin, Heparin cofactor II, Plasminogen activator inhibitor 1,Glia derived nexin/Protease nexin I, Pigment epithelium derived factor,Alpha 2-antiplasmin, Complement 1-inhibitor, 47 kDa Heat shock protein(HSP47), Neuroserpin, or Pancpin.

In another embodiment, the present invention provides that the first andthe second protease inhibitors are trypsin inhibitors such as but notlimited to: Lima bean trypsin inhibitor, Aprotinin, soy bean trypsininhibitor (SBTI), or Ovomucoid. In another, embodiment, the presentinvention provides that the first and the second protease inhibitors areLima bean trypsin inhibitor and Aprotinin. In another, embodiment, thepresent invention provides that the first and the second proteaseinhibitors are Lima bean trypsin inhibitor and soy bean trypsininhibitor (SBTI). In another, embodiment, the present invention providesthat the first and the second protease inhibitors are Lima bean trypsininhibitor, and Ovomucoid. In another, embodiment, the present inventionprovides that the first and the second protease inhibitors are Aprotininand soy bean trypsin inhibitor (SBTI). In another, embodiment, thepresent invention provides that the first and the second proteaseinhibitors are Aprotinin and Ovomucoid. In another, embodiment, thepresent invention provides that the first and the second proteaseinhibitors are soy bean trypsin inhibitor (SBTI) and Ovomucoid. Inanother, embodiment, the present invention provides that the firstprotease inhibitor is kunitz. In another, embodiment, the presentinvention provides that the second protease inhibitor is kunitz. Inanother, embodiment, the present invention provides that the firstprotease inhibitor is Bowman-Birk protease inhibitor (BBI). In another,embodiment, the present invention provides that the second proteaseinhibitor is Bowman-Birk protease inhibitor (BBI).

In another embodiment, the present invention provides that the firstprotease inhibitor is a serpine and the second protease inhibitor is aCysteine protease inhibitor. In another, embodiment, the presentinvention provides that Cysteine protease inhibitors of the inventioncomprise: cystatin, type 1 cystatins (or stefins), Cystatins of type 2,human cystatins C, D, S, SN, and SA, cystatin E/M, cystatin F, type 3cystatins, or kininogens.

In another embodiment, the present invention provides that the firstprotease inhibitor is a serpine and the second protease inhibitor is aThreonine protease inhibitor. In another, embodiment, the presentinvention provides that Threonine protease inhibitors of the inventioncomprise: Bortezomib, MLN-519, ER-807446, TMC-95A.

In another embodiment, the present invention provides that the firstprotease inhibitor is a serpine and the second protease inhibitor is anAspartic protease inhibitor. In another, embodiment, the presentinvention provides that Aspartic protease inhibitors of the inventioncomprise: α₂-Macroglobulin, Pepstatin A, Aspartic protease inhibitor 11,Aspartic protease inhibitor 1, Aspartic protease inhibitor 2, Asparticprotease inhibitor 3, Aspartic protease inhibitor 4, Aspartic proteaseinhibitor 5, Aspartic protease inhibitor 6, Aspartic protease inhibitor7, Aspartic protease inhibitor 8, Aspartic protease inhibitor 9, Pepsininhibitor Dit33, Aspartyl protease inhibitor, or Protease A inhibitor 3.

In another embodiment, the present invention provides that the firstprotease inhibitor is a serpine and the second protease inhibitor is aMetalloprotease inhibitor. In another, embodiment, the present inventionprovides that Metalloprotease inhibitors of the invention comprise:Angiotensin-1-converting enzyme inhibitory peptide, Antihemorragicfactor BJ46a, Beta-casein, Proteinase inhibitor CeKI, Venommetalloproteinase inhibitor DM43, Carboxypeptidase A inhibitor, smpI,IMPI, Alkaline proteinase, inh, Latexin, Carboxypeptidase inhibitor,Antihemorragic factor HSF, Testican-3, SPOCK3, TIMP1, Metalloproteinaseinhibitor 1, Metalloproteinase inhibitor 2, TIMP2, Metalloproteinaseinhibitor 3, TIMP3, Metalloproteinase inhibitor 4, TIMP4, Putativemetalloproteinase inhibitor tag-225, Tissue inhibitor ofmetalloprotease, WAP, kazal, immunoglobulin, or kunitz and NTRdomain-containing protein 1.

In another embodiment, the present invention provides that the firstprotease inhibitor is a Cysteine protease inhibitor and the secondprotease inhibitor is a Metalloprotease inhibitor. In anotherembodiment, the present invention provides that the first proteaseinhibitor is a Cysteine protease inhibitor and the second proteaseinhibitor is a Trypsin inhibitor. In another embodiment, the presentinvention provides that the first protease inhibitor is a Cysteineprotease inhibitor and the second protease inhibitor is a Threonineprotease inhibitor. In another embodiment, the present inventionprovides that the first protease inhibitor is a Cysteine proteaseinhibitor and the second protease inhibitor is an Asp artic proteaseinhibitor. In another embodiment, the present invention provides thatthe first protease inhibitor is a Cysteine protease inhibitor and thesecond protease inhibitor is a Metalloprotease inhibitor. In anotherembodiment, the present invention provides that the first proteaseinhibitor is a Trypsin inhibitor and the second protease inhibitor is aMetalloprotease inhibitor. In another embodiment, the present inventionprovides that the first protease inhibitor is a Trypsin inhibitor andthe second protease inhibitor is a Threonine protease inhibitor. Inanother embodiment, the present invention provides that the firstprotease inhibitor is a Trypsin inhibitor and the second proteaseinhibitor is an Aspartic protease inhibitor. In another embodiment, thepresent invention provides that the first protease inhibitor is aTrypsin inhibitor and the second protease inhibitor is a Metalloproteaseinhibitor. In another embodiment, the present invention provides thatthe first protease inhibitor is an Aspartic protease inhibitor and thesecond protease inhibitor is a Metalloprotease inhibitor. In anotherembodiment, the present invention provides that the first proteaseinhibitor is an Aspartic protease inhibitor and the second proteaseinhibitor is a Threonine protease inhibitor. In another embodiment, thepresent invention provides that the first protease inhibitor is anAspartic protease inhibitor and the second protease inhibitor is aMetalloprotease inhibitor.

In another embodiment, the present invention provides that the firstprotease inhibitor is a Cysteine protease inhibitor. In anotherembodiment, the present invention provides that the first proteaseinhibitor is a Metalloprotease inhibitor. In another embodiment, thepresent invention provides that the first protease inhibitor is aTrypsin inhibitor. In another embodiment, the present invention providesthat the first protease inhibitor is a Threonine protease inhibitor. Inanother embodiment, the present invention provides that the firstprotease inhibitor is an Aspartic protease inhibitor. In anotherembodiment, the present invention provides that the first proteaseinhibitor is a Metalloprotease inhibitor. In another embodiment, thepresent invention provides that the second protease inhibitor is aTrypsin inhibitor. In another embodiment, the present invention providesthat the second protease inhibitor is a Metalloprotease inhibitor. Inanother embodiment, the present invention provides that the secondprotease inhibitor is a Threonine protease inhibitor. In anotherembodiment, the present invention provides that the second proteaseinhibitor is an Aspartic protease inhibitor.

In some embodiments, protease inhibitors comprise suicide inhibitor,transition state inhibitor, or chelating agents. In some embodiments,the first and second protease inhibitors of the present inventioncomprise any combination of two different protease inhibitors such asbut not limited to: AEBSF-HCl, (epsilon)-aminocaproic acid, (alpha)1-antichymotypsin, antipain, antithrombin III, (alpha) 1-antitrypsin([alpha] 1-proteinase inhibitor), APMSF-HCl (4-amidinophenyl-methanesulfonyl-fluoride), sprotinin, benzamidine-HCl, chymostatin, DFP(diisopropylfluoro-phosphate), leupeptin, PEFABLOC® SC(4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride), PMSF(phenylmethyl sulfonyl fluoride), TLCK(1-Chloro-3-tosylamido-7-amino-2-heptanone HCl), TPCK(1-Chloro-3-tosylamido-4-phenyl-2-butanone), Ovomucoid, trypsininhibitor from soybean, Aprotinin, pentamidine isethionate, pepstatin,guanidium, alpha2-macroglobulin, a chelating agent of zinc, iodoacetate,zinc. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a formulation comprising a combination ofprotease inhibitors as described comprise Aprotinin and BBI. In anotherembodiment, a formulation comprising a combination of proteaseinhibitors as described comprise Aprotinin and Kunitz. In anotherembodiment, a formulation comprising a combination of proteaseinhibitors as described comprise BBI and Kunitz. In another embodiment,a formulation comprising a combination of protease inhibitors asdescribed comprise SBTI and BBI. In another embodiment, a formulationcomprising a combination of protease inhibitors as described comprisekunitz and SBTI.

In another embodiment, the amount of a first or a second proteaseinhibitor utilized in methods and compositions of the present inventionis 0.1 mg/dosage unit. In another embodiment, the amount of proteaseinhibitor is 0.2 mg/dosage unit. In another embodiment, the amount is0.3 mg/dosage unit. In another embodiment, the amount is 0.4 mg/dosageunit. In another embodiment, the amount is 0.6 mg/dosage unit. Inanother embodiment, the amount is 0.8 mg/dosage unit. In anotherembodiment, the amount is 1 mg/dosage unit. In another embodiment, theamount is 1.5 mg/dosage unit. In another embodiment, the amount is 2mg/dosage unit. In another embodiment, the amount is 2.5 mg/dosage unit.In another embodiment, the amount is 3 mg/dosage unit. In anotherembodiment, the amount is 5 mg/dosage unit. In another embodiment, theamount is 7 mg/dosage unit. In another embodiment, the amount is 10mg/dosage unit. In another embodiment, the amount is 12 mg/dosage unit.In another embodiment, the amount is 15 mg/dosage unit. In anotherembodiment, the amount is 20 mg/dosage unit. In another embodiment, theamount is 30 mg/dosage unit. In another embodiment, the amount is 50mg/dosage unit. In another embodiment, the amount is 70 mg/dosage unit.In another embodiment, the amount is 100 mg/dosage unit.

In another embodiment, the amount of a first or a second proteaseinhibitor is 0.1-1 mg/dosage unit. In another embodiment, the amount ofprotease inhibitor is 0.2-1 mg/dosage unit. In another embodiment, theamount is 0.3-1 mg/dosage unit. In another embodiment, the amount is0.5-1 mg/dosage unit. In another embodiment, the amount is 0.1-2mg/dosage unit. In another embodiment, the amount is 0.2-2 mg/dosageunit. In another embodiment, the amount is 0.3-2 mg/dosage unit. Inanother embodiment, the amount is 0.5-2 mg/dosage unit. In anotherembodiment, the amount is 1-2 mg/dosage unit. In another embodiment, theamount is 1-10 mg/dosage unit. In another embodiment, the amount is 2-10mg/dosage unit. In another embodiment, the amount is 3-10 mg/dosageunit. In another embodiment, the amount is 5-10 mg/dosage unit. Inanother embodiment, the amount is 1-20 mg/dosage unit. In anotherembodiment, the amount is 2-20 mg/dosage unit. In another embodiment,the amount is 3-20 mg/dosage unit. In another embodiment, the amount is5-20 mg/dosage unit. In another embodiment, the amount is 10-20mg/dosage unit. In another embodiment, the amount is 10-100 mg/dosageunit. In another embodiment, the amount is 20-100 mg/dosage unit. Inanother embodiment, the amount is 30-100 mg/dosage unit. In anotherembodiment, the amount is 50-100 mg/dosage unit. In another embodiment,the amount is 10-200 mg/dosage unit. In another embodiment, the amountis 20-200 mg/dosage unit. In another embodiment, the amount is 30-200mg/dosage unit. In another embodiment, the amount is 50-200 mg/dosageunit. In another embodiment, the amount is 100-200 mg/dosage unit.

In another embodiment, the amount of a first or a second proteaseinhibitor utilized in methods and compositions of the present inventionis 1000 k.i.u. (kallikrein inactivator units)/pill. In anotherembodiment, the amount is 10 k.i.u./dosage unit. In another embodiment,the amount is 12 k.i.u./dosage unit. In another embodiment, the amountis 15 k.i.u./dosage unit. In another embodiment, the amount is 20UAL/dosage unit. In another embodiment, the amount is 30 k.i.u./dosageunit. In another embodiment, the amount is 40 k.i.u./dosage unit. Inanother embodiment, the amount is 50 k.i.u./dosage unit. In anotherembodiment, the amount is 70 k.i.u./dosage unit. In another embodiment,the amount is 100 k.i.u./dosage unit. In another embodiment, the amountis 150 k.i.u./dosage unit. In another embodiment, the amount is 200UAL/dosage unit. In another embodiment, the amount is 300 k.i.u./dosageunit. In another embodiment, the amount is 500 k.i.u./dosage unit. Inanother embodiment, the amount is 700 k.i.u./dosage unit. In anotherembodiment, the amount is 1500 k.i.u./dosage unit. In anotherembodiment, the amount is 3000 k.i.u./dosage unit. In anotherembodiment, the amount is 4000 k.i.u./dosage unit. In anotherembodiment, the amount is 5000 k.i.u./dosage unit. Each amount of afirst or a second protease inhibitor represents a separate embodiment ofthe present invention.

In some embodiments, omega-3 fatty acid can be found in vegetablesources such as the seeds of chia, perilla, flax, walnuts, purslane,lingonberry, seabuckthom, and hemp. In some embodiments, omega-3 fattyacids can also be found in the fruit of the acai palm. In anotherembodiment, the omega-3 fatty acid has been provided in the form of asynthetic omega-3 fatty acid. In one embodiment, the omega-3 fatty acidof methods and compositions of the present invention has been providedto the composition in the form of a fish oil. In another embodiment, theomega-3 fatty acid has been provided in the form of canola oil. Inanother embodiment, the omega-3 fatty acid has been provided in the formof flaxseed oil. In another embodiment, the omega-3 fatty acid has beenprovided in the form of any other omega-3 fatty acid-rich source knownin the art. In another embodiment, the omega-3 fatty acid has beenprovided in the form of a synthetic omega-3 fatty acid. Each form ofomega-3 fatty acids represents a separate embodiment of the presentinvention.

In another embodiment, the omega-3 fatty acid of methods andcompositions of the present invention is an omega-3 polyunsaturatedfatty acid. In another embodiment, the omega-3 fatty acid is DHA, anomega-3, polyunsaturated, 22-carbon fatty acid also referred to as4,7,10,13,16,19-docosahexaenoic acid. In another embodiment, the omega-3fatty acid is □-linolenic acid (9,12,15-octadecatrienoic acid). Inanother embodiment, the omega-3 fatty acid is stearidonic acid(6,9,12,15-octadecatetraenoic acid). In another embodiment, the omega-3fatty acid is eicosatrienoic acid (ETA; 11,14,17-eicosatrienoic acid).In another embodiment, the omega-3 fatty acid is eicsoatetraenoic acid(8,11,14,17-eicosatetraenoic acid). In one embodiment, the omega-3 fattyacid is eicosapentaenoic acid (EPA; 5,8,11,14,17-eicosapentaenoic acid).In another embodiment, the omega-3 fatty acid is eicosahexaenoic acid(also referred to as “EPA”; 5,7,9,11,14,17-eicosahexaenoic acid). Inanother embodiment, the omega-3 fatty acid is docosapentaenoic acid(DPA; 7, 10, 13, 16, 19-docosapenatenoic acid). In another embodiment,the omega-3 fatty acid is tetracosahexaenoic acid (6, 9, 12, 15, 18,21-tetracosahexaenoic acid). In another embodiment, the omega-3 fattyacid is any other omega-3 fatty acid known in the art. Each omega-3fatty acid represents a separate embodiment of the present invention.

In another embodiment, compositions of the present invention furthercomprise a substance that enhances absorption of a protein of theinvention through an intestinal mucosal barrier. In another embodiment,compositions of the present invention further comprise a substance thatenhances absorption of insulin through an intestinal mucosal barrier. Inanother embodiment, compositions of the present invention furthercomprise a substance that enhances absorption of Exenatide through anintestinal mucosal barrier. In another embodiment, compositions of thepresent invention further comprise a substance that reduces thedegradation of Exenatide in the digestive system. In another embodiment,compositions of the present invention further comprise a substance thatreduces the degradation of Exenatide in the stomach. In anotherembodiment, compositions of the present invention further comprise asubstance that reduces the degradation of Exenatide in the intestine.Such a substance is referred to herein as an “enhancer.” As providedherein, enhancers, when used together with omega-3 fatty acids orprotease inhibitors, enhance the ability of a protein to be absorbed inthe intestine. As provided herein, enhancers, when used together withomega-3 fatty acids and/or protease inhibitors, enhance the ability ofinsulin to be absorbed in the intestine. As provided herein, enhancers,when used together with omega-3 fatty acids and/or protease inhibitors,enhance the ability of Exenatide to be absorbed in the intestine.

In one embodiment, the enhancer is didecanoylphosphatidylcholine (DDPC).In one embodiment, the enhancer is a chelating agent such asethylenediaminetetraacetic acid (EDTA) or egtazic acid EGTA. In anotherembodiment, EDTA is sodium-EDTA. In some embodiments, the enhancer is NOdonor. In some embodiments, the enhancer is a bile acid,glycine-conjugated form of a bile acid, or an alkali metal salt. In oneembodiment, absorption enhancement is achieved through utilization of acombination of α-galactosidase and β-mannanase. In some embodiments, theenhancer is a fatty acid such as sodium caprate. In one embodiment, theenhancer is sodium glycocholate. In one embodiment, the enhancer issodium salicylate. In one embodiment, the enhancer isn-dodecyl-β-D-maltopyranoside. In some embodiments, surfactants serve asabsorption enhancer. In one embodiment, the enhancer is chitisan such asN, N,N-trimethyl chitosan chloride (TMC).

In one embodiment, NO donors of the present invention comprise3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine,N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, orS-Nitroso-N-acetylpenicillamine

In another embodiment, the bile acid is cholic acid. In anotherembodiment, the bile acid is chenodeoxycholic acid. In anotherembodiment, the bile acid is taurocholic acid. In another embodiment,the bile acid is taurochenodeoxycholic acid. In another embodiment, thebile acid is glycocholic acid. In another embodiment, the bile acid isglycochenocholic acid. In another embodiment, the bile acid is 3beta-monohydroxychloric acid. In another embodiment, the bile acid islithocholic acid. In another embodiment, the bile acid is 5beta-cholanic acid. In another embodiment, the bile acid is3,12-diol-7-one-5 beta-cholanic acid. In another embodiment, the bileacid is 3 alpha-hydroxy-12-ketocholic acid. In another embodiment, thebile acid is 3 beta-hydroxy-12-ketocholic acid. In another embodiment,the bile acid is 12 alpha-3 beta-dihydrocholic acid. In anotherembodiment, the bile acid is ursodesoxycholic acid.

In one embodiment, the enhancer is a nonionic surfactant. In oneembodiment, the enhancer is a nonionic polyoxyethylene ether surfaceactive agent (e.g one having an HLB value of 6 to 19, wherein theaverage number of polyoxyethylene units is 4 to 30). In anotherembodiment, the enhancer is an anionic surface active agents. In anotherembodiment, the enhancer is a cationic surface active agent. In anotherembodiment, the enhancer is an ampholytic surface active agent. In oneembodiment, zwitteruionic surfactants such as acylcarnitines serve asabsorption enhancers.

In another embodiment, the amount of enhancer utilized in methods andcompositions of the present invention is 0.1 mg/dosage unit. In anotherembodiment, the amount of enhancer is 0.2 mg/dosage unit. In anotherembodiment, the amount is 0.3 mg/dosage unit. In another embodiment, theamount is 0.4 mg/dosage unit. In another embodiment, the amount is 0.6mg/dosage unit. In another embodiment, the amount is 0.8 mg/dosage unit.In another embodiment, the amount is 1 mg/dosage unit. In anotherembodiment, the amount is 1.5 mg/dosage unit. In another embodiment, theamount is 2 mg/dosage unit. In another embodiment, the amount is 2.5mg/dosage unit. In another embodiment, the amount is 3 mg/dosage unit.In another embodiment, the amount is 5 mg/dosage unit. In anotherembodiment, the amount is 7 mg/dosage unit. In another embodiment, theamount is 10 mg/dosage unit. In another embodiment, the amount is 12mg/dosage unit. In another embodiment, the amount is 15 mg/dosage unit.In another embodiment, the amount is 20 mg/dosage unit. In anotherembodiment, the amount is 30 mg/dosage unit. In another embodiment, theamount is 50 mg/dosage unit. In another embodiment, the amount is 70mg/dosage unit. In another embodiment, the amount is 100 mg/dosage unit.

In another embodiment, the amount of enhancer is 0.1-1 mg/dosage unit.In another embodiment, the amount of enhancer is 0.2-1 mg/dosage unit.In another embodiment, the amount is 0.3-1 mg/dosage unit. In anotherembodiment, the amount is 0.5-1 mg/dosage unit. In another embodiment,the amount is 0.1-2 mg/dosage unit. In another embodiment, the amount is0.2-2 mg/dosage unit. In another embodiment, the amount is 0.3-2mg/dosage unit. In another embodiment, the amount is 0.5-2 mg/dosageunit. In another embodiment, the amount is 1-2 mg/dosage unit. Inanother embodiment, the amount is 1-10 mg/dosage unit. In anotherembodiment, the amount is 2-10 mg/dosage unit. In another embodiment,the amount is 3-10 mg/dosage unit. In another embodiment, the amount is5-10 mg/dosage unit. In another embodiment, the amount is 1-20 mg/dosageunit. In another embodiment, the amount is 2-20 mg/dosage unit. Inanother embodiment, the amount is 3-20 mg/dosage unit. In anotherembodiment, the amount is 5-20 mg/dosage unit. In another embodiment,the amount is 10-20 mg/dosage unit. In another embodiment, the amount is10-100 mg/dosage unit. In another embodiment, the amount is 20-100mg/dosage unit. In another embodiment, the amount is 30-100 mg/dosageunit. In another embodiment, the amount is 50-100 mg/dosage unit. Inanother embodiment, the amount is 10-200 mg/dosage unit. In anotherembodiment, the amount is 20-200 mg/dosage unit. In another embodiment,the amount is 30-200 mg/dosage unit. In another embodiment, the amountis 50-200 mg/dosage unit. In another embodiment, the amount is 100-200mg/dosage unit. Each type and amount of enhancer represents a separateembodiment of the present invention.

In another embodiment, compositions of the present invention furthercomprise a coating that inhibits digestion of the composition in thestomach of a subject. In one embodiment, coating inhibits digestion ofthe composition in the stomach of a subject. In one embodiment, thecoated dosage forms of the present invention release drug when pH movetowards alkaline range. In one embodiment, coating is a monolayer,wherein in other embodiments coating applied in multilayers. In oneembodiment, coating is a bioadhesive polymer that selectively binds theintestinal mucosa and thus enables drug release in the attachment site.In one embodiment, the enteric coating is an enteric film coating. Insome embodiment, coating comprises biodegradable polysaccharide,chitosan, aquateric aqueous, aquacoat ECD, azo polymer, celluloseacetate phthalate, cellulose acetate trimelliate, hydroxypropylmethylcellulose phthalate, gelatin, poly vinyl acetate phthalate, hydrogel,pulsincap, or a combination thereof. In one embodiment, pH sensitivecoating will be used according to the desired release site and/orprofile as known to one skilled in the art.

In one embodiment, the coating is an enteric coating. Methods forenteric coating are well known in the art, and are described, forexample, in Siepmann F, Siepmann J et al, Blends of aqueous polymerdispersions used for pellet coating: importance of the particle size. JControl Release 2005; 105(3): 226-39; and Huyghebaert N, Vermeire A,Remon J P. In vitro evaluation of coating polymers for enteric coatingand human ileal targeting. Int J Pharm 2005; 298(1): 26-37. Each methodrepresents a separate embodiment of the present invention.

In another embodiment, Eudragit®, an acrylic polymer, is used as theenteric coating. The use of acrylic polymers for the coating ofpharmaceutical preparations is well known in the art. Eudragit AcrylicPolymers have been shown to be safe, and are neither absorbed normetabolized by the body, but rather are eliminated.

In another embodiment, the coating is a gelatin coating. In anotherembodiment, microencapsulation is used to protect the insulin againstdecomposition in the stomach. In another embodiment, the coating is agelatin coating. In another embodiment, microencapsulation is used toprotect Exenatide against decomposition in the stomach. Methods forapplying a gelatin coating and for microencapsulation are well known inthe art. Each method represents a separate embodiment of the presentinvention.

In another embodiment, the coating is a film-coating. In anotherembodiment, the coating is ethylcellulose. In another embodiment, thecoating is a water-based dispersion of ethylcellulose, e.g.hydroxypropylmethylcelullose (HPMC) E15. In another embodiment, thecoating is a gastro-resistant coatings, e.g. a polymer containingcarboxylic acid groups as a functional moiety. In another embodiment,the coating is a monolithic matrix. In another embodiment, the coatingis a cellulose ether (e.g. hypromellose (HPMC). Each type of coatingrepresents a separate embodiment of the present invention.

In one embodiment, the protein is a recombinant protein. In oneembodiment, the protein is an insulin. In another embodiment, theprotein is a glucagon. In another embodiment, the protein is aninterferon gamma. In another embodiment, the protein is an interferonalpha. In another embodiment, the protein is a growth hormone. Inanother embodiment, the protein is an erythropoietin. In anotherembodiment, the protein is Exenatide. In another embodiment, the proteinis granulocyte colony stimulating factor (G-CSF). In another embodiment,the protein is any other protein known in the art.

In another embodiment, the protein is a growth hormone. In oneembodiment, the growth hormone is somatotropin. In another embodiment,the growth hormone is Insulin Growth Factor-I (IGF-I). In anotherembodiment, the growth hormone is any other growth hormone known in theart.

The molecular weights of some of the proteins mentioned above are asfollows: insulin-6 kilodalton (kDa); glucagon-3.5 kDa; interferon, 28kDa, growth hormone-21.5-47 kDa; human serum albumin-69 kDa;erythropoietin-34 kDa; G-CSF-30-34 kDa. Thus, in one embodiment, themolecular weight of these proteins is appropriate for administration bymethods of the present invention.

In another embodiment, methods and compositions of the present inventionare used to administer a human serum albumin. Human serum albumin isnot, in one embodiment, considered to be a pharmaceutically-activecomponent; however, it can be used in the context of the presentinvention as a therapeutically-beneficial carrier for an activecomponent. Each type of protein represents a separate embodiment of thepresent invention.

In one embodiment, the protein is an enzyme. In some embodiments, theprotein is a receptor ligand, transporter, or a storage protein. In oneembodiment, the protein is a structural protein.

In some embodiments, the enzyme is an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase. In some embodiments,oxidoreductases act on the aldehyde or oxo group of donors, on the CH—CHgroup of donors, on the CH—NH(2) group of donors, on the CH—NH group ofdonors, on NADH or NADPH, on the CH—OH group of donors, on nitrogenouscompounds as donors, on a sulfur group of donors, on a heme group ofdonors, on diphenols and related substances as donors, on a peroxide asacceptor, on hydrogen as donor, on single donors with incorporation ofmolecular oxygen, on paired donors, on superoxide as acceptor, oxidizingmetal ions, on CH or CH(2) groups, on iron-sulfur proteins as donors, onreduced flavodoxin as donor, on phosphorus or arsenic in donors, or onx-H and y-H to form an x-y bond.

In some embodiments, transferases are acyltransferases orglycosyltransferases. In some embodiments, transferases transferaldehyde or ketone residues. In some embodiments, transferases transferalkyl or aryl groups, other than methyl groups. In some embodiments,transferases transfer nitrogenous, phosphorous, sulfur or seleniumcontaining groups.

In some embodiments, hydrolases are glycosylases or act on ether bonds,on peptide bonds, on carbon-nitrogen bonds, other than peptide bonds, onacid anhydrides, on carbon-carbon bonds, on halide bonds, onphosphorus-nitrogen bonds, on sulfur-nitrogen bonds, oncarbon-phosphorus bonds, on sulfur-sulfur bonds, or on carbon-sulfurbonds.

In some embodiments, lyases are carbon-carbon lyases, carbon-oxygenlyases, carbon-nitrogen lyases, carbon-sulfur lyases, carbon-halidelyases, phosphorus-oxygen lyases, or other lyases.

In some embodiments, isomerases are racemases or epimerases,cis-trans-isomerases, intramolecular oxidoreductases, intramoleculartransferases, intramolecular lyases, or other isomerases.

In some embodiments, ligases form carbon-sulfur bonds, carbon-nitrogenbonds, carbon-carbon bonds, phosphoric ester bonds, or nitrogen-metalbonds.

In some embodiments, transporter proteins are annexins, ATP-bindingcassette transporters, hemoglobin, ATPases, calcium channels, potassiumchannels, sodium channels, or solute carriers.

In some embodiments, storage proteins comprise albumins, lactoglobulins,casein ovomucin, ferritin, phosvitin, lactoferrin, or vitellogenin. Inone embodiment, albumins comprise avidin, ovalbumin, serum albumin,parvalbumin, c-reactive protein prealbumin, conalbumin, ricin,lactalbumin, methemalbumin, or transthyretin.

In some embodiments, structural proteins comprise amyloid, collagenelastin, or fibrillin.

In some embodiments, the protein is a viral protein, bacterial protein,invertebrate protein, or vertebrate protein. In some embodiments, theprotein is a recombinant protein. In one embodiment, the protein is arecombinant protein. In one embodiment, the recombinant protein is arecombinant human protein.

In one embodiment, the present invention provides a compositioncomprising an insulin protein and at least two protease inhibitors. Inone embodiment, the present invention provides a composition comprisinga Exenatide and at least two protease inhibitors. In one embodiment, thepresent invention provides a composition comprising an insulin proteinand two protease inhibitors. In one embodiment, the present inventionprovides a composition comprising a Exenatide and two proteaseinhibitors. In one embodiment, the present invention provides acomposition comprising a Exenatide and 3 protease inhibitors. In oneembodiment, the present invention provides a composition comprising aninsulin protein and 4 protease inhibitors. In one embodiment, thepresent invention provides a composition comprising a Exenatide and 4protease inhibitors. In one embodiment, the present invention provides acomposition comprising an insulin protein and 5 protease inhibitors. Inone embodiment, the present invention provides a composition comprisinga Exenatide and 5 protease inhibitors. In one embodiment, the presentinvention provides a composition comprising an insulin protein and atleast 5 protease inhibitors. In one embodiment, the present inventionprovides a composition comprising an insulin protein and at least 7protease inhibitors. In one embodiment, the present invention provides acomposition comprising an insulin protein and at least 10 proteaseinhibitors.

In another embodiment, the present invention provides a compositioncomprising an active protein of the invention, at least two proteaseinhibitors, and an omega-3 fatty acid. In another embodiment, thepresent invention provides a composition comprising an active protein ofthe invention, at least two protease inhibitors, EDTA or a salt thereof(such as Na-EDTA), and an omega-3 fatty acid.

In another embodiment, the present invention provides that the use oftwo protease inhibitors in a single oral composition dramatically,unexpectedly, increase the bio availability of a protein of theinvention. In another embodiment, the present invention provides thatthe use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin. Inanother embodiment, the present invention provides that the use of twoprotease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide. In anotherembodiment, the present invention provides that the use of two serpinsin a single oral composition dramatically, unexpectedly, increase thebioavailability of a protein of the invention. In another embodiment,the present invention provides that the use of two serpins in a singleoral composition dramatically, unexpectedly, increase thebioavailability of insulin. In another embodiment, the present inventionprovides that the use of two serpins in a single oral compositiondramatically, unexpectedly, increase the bio availability of Exenatide.In another embodiment, the present invention provides that the use oftwo trypsin inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of a protein of theinvention. In another embodiment, the present invention provides thatthe use of two trypsin inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin. Inanother embodiment, the present invention provides that the use of twotrypsin inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide. In anotherembodiment, the present invention provides that the use of SBTI andAprotinin in a single oral composition dramatically, unexpectedly,increase the bioavailability of a protein of the invention. In anotherembodiment, the present invention provides that the use of SBTI andAprotinin in a single oral composition dramatically, unexpectedly,increase the bioavailability of insulin. In another embodiment, thepresent invention provides that the use of SBTI and Aprotinin in asingle oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide.

In another embodiment, the present invention provides that the use of aserpin and a Cysteine protease inhibitor in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention. In another embodiment, the present invention providesthat the use of a serpin and a Cysteine protease inhibitor in a singleoral composition dramatically, unexpectedly, increase thebioavailability of insulin. In another embodiment, the present inventionprovides that the use of a serpin and a Cysteine protease inhibitor in asingle oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide. In another embodiment, the presentinvention provides that the use of a serpin and a Threonine proteaseinhibitor in a single oral composition dramatically, unexpectedly,increase the bioavailability of a protein of the invention. In anotherembodiment, the present invention provides that the use of a serpin anda Threonine protease inhibitor in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin. Inanother embodiment, the present invention provides that the use of aserpin and a Threonine protease inhibitor in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide.In another embodiment, the present invention provides that the use of aserpin and a Metalloprotease inhibitor in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention. In another embodiment, the present invention providesthat the use of a serpin and a Metalloprotease inhibitor in a singleoral composition dramatically, unexpectedly, increase thebioavailability of insulin. In another embodiment, the present inventionprovides that the use of a serpin and a Metalloprotease inhibitor in asingle oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide. In another embodiment, the presentinvention provides that the use of a serpin and an Aspartic proteaseinhibitor in a single oral composition dramatically, unexpectedly,increase the bio availability of a protein of the invention. In anotherembodiment, the present invention provides that the use of a serpin andan Aspartic protease inhibitor in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin. Inanother embodiment, the present invention provides that the use of aserpin and an Aspartic protease inhibitor in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide.

In another embodiment, the present invention provides that the use oftwo protease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of a protein of theinvention. In another embodiment, the present invention provides thatthe use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin. Inanother embodiment, the present invention provides that the use of twoprotease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide. In anotherembodiment, the present invention provides that the use of two proteaseinhibitors in a single oral composition dramatically, unexpectedly,increase the bioavailability of insulin in a human subject. In anotherembodiment, the present invention provides that the use of two proteaseinhibitors in a single oral composition dramatically, unexpectedly,increase the bioavailability of Exenatide in a human subject. In anotherembodiment, the present invention provides that the use of two proteaseinhibitors in a single oral composition dramatically, unexpectedly,increase the bioavailability of a protein of the invention in a humansubject.

In another embodiment, the present invention provides that the use oftwo protease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin in a human subjectby at least 10%. In another embodiment, the present invention providesthat the use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide ina human subject by at least 10%. In another embodiment, the presentinvention provides that the use of two protease inhibitors in a singleoral composition dramatically, unexpectedly, increase the bioavailability of insulin in a human subject by at least 20%. In anotherembodiment, the present invention provides that the use of two proteaseinhibitors in a single oral composition dramatically, unexpectedly,increase the bioavailability of Exenatide in a human subject by at least20%. In another embodiment, the present invention provides that the useof two protease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin in a human subjectby at least 30%. In another embodiment, the present invention providesthat the use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide ina human subject by at least 30%. In another embodiment, the presentinvention provides that the use of two protease inhibitors in a singleoral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 40%. Inanother embodiment, the present invention provides that the use of twoprotease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide in a humansubject by at least 40%. In another embodiment, the present inventionprovides that the use of two protease inhibitors in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofinsulin in a human subject by at least 50%. In another embodiment, thepresent invention provides that the use of two protease inhibitors in asingle oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 50%. Inanother embodiment, the present invention provides that the use of twoprotease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin in a human subjectby at least 60%. In another embodiment, the present invention providesthat the use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide ina human subject by at least 60%. In another embodiment, the presentinvention provides that the use of two protease inhibitors in a singleoral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 70%. Inanother embodiment, the present invention provides that the use of twoprotease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide in a humansubject by at least 70%. In another embodiment, the present inventionprovides that the use of two protease inhibitors in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofinsulin in a human subject by at least 80%. In another embodiment, thepresent invention provides that the use of two protease inhibitors in asingle oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 80%. Inanother embodiment, the present invention provides that the use of twoprotease inhibitors in a single oral composition dramatically,unexpectedly, increase the bio availability of insulin in a humansubject by at least 90%. In another embodiment, the present inventionprovides that the use of two protease inhibitors in a single oralcomposition dramatically, unexpectedly, increase the bio availability ofExenatide in a human subject by at least 90%. In another embodiment, thepresent invention provides that the use of two protease inhibitors in asingle oral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 100%. Inanother embodiment, the present invention provides that the use of twoprotease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide in a humansubject by at least 100%.

In another embodiment, the present invention provides that the use ofAprotinin and SBTI in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin in a human subjectby at least 10%. In another embodiment, the present invention providesthat the use of Aprotinin and SBTI in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 20%. In another embodiment, the presentinvention provides that the use of Aprotinin and SBTI in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofinsulin in a human subject by at least 30%. In another embodiment, thepresent invention provides that the use of Aprotinin and SBTI in asingle oral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 40%. Inanother embodiment, the present invention provides that the use ofAprotinin and SBTI in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin in a human subjectby at least 50%. In another embodiment, the present invention providesthat the use of Aprotinin and SBTI in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 60%. In another embodiment, the presentinvention provides that the use of Aprotinin and SBTI in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofinsulin in a human subject by at least 70%. In another embodiment, thepresent invention provides that the use of Aprotinin and SBTI in asingle oral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 80%. Inanother embodiment, the present invention provides that the use ofAprotinin and SBTI in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin in a human subjectby at least 90%. In another embodiment, the present invention providesthat the use of Aprotinin and SBTI in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 100%.

In another embodiment, the present invention provides that the use ofAprotinin and SBTI in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide in a humansubject by at least 10%. In another embodiment, the present inventionprovides that the use of Aprotinin and SBTI in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide ina human subject by at least 20%. In another embodiment, the presentinvention provides that the use of Aprotinin and SBTI in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofExenatide in a human subject by at least 30%. In another embodiment, thepresent invention provides that the use of Aprotinin and SBTI in asingle oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 40%. Inanother embodiment, the present invention provides that the use ofAprotinin and SBTI in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide in a humansubject by at least 50%. In another embodiment, the present inventionprovides that the use of Aprotinin and SBTI in a single oral compositiondramatically, unexpectedly, increase the bio availability of Exenatidein a human subject by at least 60%. In another embodiment, the presentinvention provides that the use of Aprotinin and SBTI in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofExenatide in a human subject by at least 70%. In another embodiment, thepresent invention provides that the use of Aprotinin and SBTI in asingle oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 80%. Inanother embodiment, the present invention provides that the use ofAprotinin and SBTI in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide in a humansubject by at least 90%. In another embodiment, the present inventionprovides that the use of Aprotinin and SBTI in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide ina human subject by at least 100%.

In another embodiment, the present invention provides that the use oftwo protease inhibitors in a single oral composition dramatically,unexpectedly, increase the bioavailability of a protein of the inventionin a human subject by at least 10%. In another embodiment, the presentinvention provides that the use of two protease inhibitors in a singleoral composition dramatically, unexpectedly, increase thebioavailability of a protein of the invention in a human subject by atleast 20%. In another embodiment, the present invention provides thatthe use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention in a human subject by at least 30%. In another embodiment,the present invention provides that the use of two protease inhibitorsin a single oral composition dramatically, unexpectedly, increase thebioavailability of a protein of the invention in a human subject by atleast 40%. In another embodiment, the present invention provides thatthe use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention in a human subject by at least 50%. In another embodiment,the present invention provides that the use of two protease inhibitorsin a single oral composition dramatically, unexpectedly, increase thebioavailability of a protein of the invention in a human subject by atleast 60%. In another embodiment, the present invention provides thatthe use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention in a human subject by at least 70%. In another embodiment,the present invention provides that the use of two protease inhibitorsin a single oral composition dramatically, unexpectedly, increase thebioavailability of a protein of the invention in a human subject by atleast 80%. In another embodiment, the present invention provides thatthe use of two protease inhibitors in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention in a human subject by at least 90%. In another embodiment,the present invention provides that the use of two protease inhibitorsin a single oral composition dramatically, unexpectedly, increase thebioavailability of a protein of the invention in a human subject by atleast 100%.

In another embodiment, this invention further provides the use ofsustained release dosage forms (e.g. sustained releasemicroencapsulation) that enable the treatment frequency to be reduced toonce or twice a day. In another embodiment, the insulin dosage isincreased correspondingly with decreasing frequency of administration.In another embodiment, the Exenatide dosage is increased correspondinglywith decreasing frequency of administration. Each type of coating,dosage form, etc, that inhibits digestion of the composition in thestomach represents a separate embodiment of the present invention.

Methods of measuring insulin levels are well known in the art. In oneembodiment, levels of recombinant insulin are measuring using a humaninsulin radio-immunoassay (RIA) kit, e.g. the kit manufactured by. LincoResearch Inc, (St. Charles, Mo.). In another embodiment, levels of Cpeptide are measured as well, to determine the relative contributions ofendogenous and exogenous insulin to observed rises in insulin levels. Inanother embodiment, insulin ELISA kits are used. In another embodiment,insulin levels are measured by any other method known in the art. Inanother embodiment, Exenatide levels are measured by a method known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a multiparticulate dosage forms is used toinhibit digestion of the composition in the stomach. In anotherembodiment, a multiparticulate dosage forms is used to inhibit digestionof the composition in the stomach. In another embodiment, the presentinvention comprises administering separate dosage forms wherein eachprotease inhibitor is in a separate dosage form and the protein is in anadditional dosage form. In another embodiment, the present inventioncomprises administering separate dosage forms wherein the two proteaseinhibitors are in a single dosage form and the protein is in anadditional dosage form.

In another embodiment, the present invention provides a method for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising the protein and at least two protease inhibitors,thereby orally administering a protein with an enzymatic activity to asubject. In another embodiment, the present invention provides a methodfor oral administration of a protein with an enzymatic activity to asubject, whereby a substantial fraction of the protein retains theenzymatic activity after absorption through an intestinal mucosal batherof the subject, comprising administering orally to the subject apharmaceutical composition comprising the protein and at least twoprotease inhibitors, thereby orally administering a protein with anenzymatic activity to a human subject. In another embodiment, thepresent invention provides a method for oral administration of insulinto a subject, whereby a substantial fraction of insulin retains itsactivity after absorption through an intestinal mucosal bather of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising insulin and at least two protease inhibitors,thereby orally administering a protein with an enzymatic activity to asubject. In another embodiment, the present invention provides a methodfor oral administration of Exenatide to a subject, whereby a substantialfraction of Exenatide retains its activity after absorption through anintestinal mucosal barrier of the subject, comprising administeringorally to the subject a pharmaceutical composition comprising Exenatideand at least two protease inhibitors, thereby orally administering aprotein with an enzymatic activity to a subject.

In another embodiment, the present invention provides a method for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising the protein at least two protease inhibitors andan omega-3 fatty acid, thereby orally administering a protein with anenzymatic activity to a subject. In another embodiment, the presentinvention provides a method for oral administration of a protein with anenzymatic activity to a subject, whereby a substantial fraction of theprotein retains the enzymatic activity after absorption through anintestinal mucosal barrier of the subject, comprising administeringorally to the subject a pharmaceutical composition comprising theprotein, at least two protease inhibitors and an omega-3 fatty acid,thereby orally administering a protein with an enzymatic activity to ahuman subject. In another embodiment, the present invention provides amethod for oral administration of insulin to a subject, whereby asubstantial fraction of insulin retains its activity after absorptionthrough an intestinal mucosal bather of the subject, comprisingadministering orally to the subject a pharmaceutical compositioncomprising insulin, at least two protease inhibitors and an omega-3fatty acid, thereby orally administering a protein with an enzymaticactivity to a subject. In another embodiment, the present inventionprovides a method for oral administration of Exenatide to a subject,whereby a substantial fraction of Exenatide retains its activity afterabsorption through an intestinal mucosal barrier of the subject,comprising administering orally to the subject a pharmaceuticalcomposition comprising Exenatide, at, least two protease inhibitors andan omega-3 fatty acid, thereby orally administering a protein with anenzymatic activity to a subject.

In another embodiment, the present invention provides a method for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising the protein at least two protease inhibitors, anomega-3 fatty acid, and Na-EDTA, thereby orally administering a proteinwith an enzymatic activity to a subject. In another embodiment, thepresent invention provides a method for oral administration of a proteinwith an enzymatic activity to a subject, whereby a substantial fractionof the protein retains the enzymatic activity after absorption throughan intestinal mucosal bather of the subject, comprising administeringorally to the subject a pharmaceutical composition comprising theprotein, at least two protease inhibitors, an omega-3 fatty acid, andNa-EDTA, thereby orally administering a protein with an enzymaticactivity to a human subject. In another embodiment, the presentinvention provides a method for oral administration of insulin to asubject, whereby a substantial fraction of insulin retains its activityafter absorption through an intestinal mucosal barrier of the subject,comprising administering orally to the subject a pharmaceuticalcomposition comprising insulin, at least two protease inhibitors, anomega-3 fatty acid, and Na-EDTA, thereby orally administering a proteinwith an enzymatic activity to a subject. In another embodiment, thepresent invention provides a method for oral administration of Exenatideto a subject, whereby a substantial fraction of Exenatide retains itsactivity after absorption through an intestinal mucosal bather of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising Exenatide, at least two protease inhibitors, anomega-3 fatty acid, and Na-EDTA, thereby orally administering a proteinwith an enzymatic activity to a subject.

In another embodiment, the present invention provides a method for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising the protein at least two protease inhibitors andNa-EDTA, thereby orally administering a protein with an enzymaticactivity to a subject. In another embodiment, the present inventionprovides a method for oral administration of a protein with an enzymaticactivity to a subject, whereby a substantial fraction of the proteinretains the enzymatic activity after absorption through an intestinalmucosal bather of the subject, comprising administering orally to thesubject a pharmaceutical composition comprising the protein, at leasttwo protease inhibitors and Na-EDTA, thereby orally administering aprotein with an enzymatic activity to a human subject. In anotherembodiment, the present invention provides a method for oraladministration of insulin to a subject, whereby a substantial fractionof insulin retains its activity after absorption through an intestinalmucosal bather of the subject, comprising administering orally to thesubject a pharmaceutical composition comprising insulin, at least twoprotease inhibitors and Na-EDTA, thereby orally administering a proteinwith an enzymatic activity to a subject. In another embodiment, thepresent invention provides a method for oral administration of Exenatideto a subject, whereby a substantial fraction of Exenatide retains itsactivity after absorption through an intestinal mucosal bather of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising Exenatide, at least two protease inhibitors andNa-EDTA, thereby orally administering a protein with an enzymaticactivity to a subject.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising an insulinand at least two protease inhibitors, thereby treating diabetesmellitus. In another embodiment, the present invention provides a methodfor treating diabetes mellitus in a human subject, comprisingadministering orally to the subject a pharmaceutical compositioncomprising an insulin and at least two protease inhibitors, therebytreating diabetes mellitus.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising aninsulin, omega-3 fatty acid, and at least two protease inhibitors,thereby treating diabetes mellitus. In another embodiment, the presentinvention provides a method for treating diabetes mellitus in a humansubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising an insulin, omega-3 fatty acid, and at least twoprotease inhibitors, thereby treating diabetes mellitus.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising aninsulin, Na-EDTA, omega-3 fatty acid, and at least two proteaseinhibitors, thereby treating diabetes mellitus. In another embodiment,the present invention provides a method for treating diabetes mellitusin a human subject, comprising administering orally to the subject apharmaceutical composition comprising an insulin, Na-EDTA, omega-3 fattyacid, and at least two protease inhibitors, thereby treating diabetesmellitus.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising aExenatide and at least two protease inhibitors, thereby treatingdiabetes mellitus. In another embodiment, the present invention providesa method for treating diabetes mellitus in a human subject, comprisingadministering orally to the subject a pharmaceutical compositioncomprising a Exenatide and at least two protease inhibitors, therebytreating diabetes mellitus.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising aExenatide, omega-3 fatty acid, and at least two protease inhibitors,thereby treating diabetes mellitus. In another embodiment, the presentinvention provides a method for treating diabetes mellitus in a humansubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising a Exenatide, omega-3 fatty acid, and at least twoprotease inhibitors, thereby treating diabetes mellitus.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising aExenatide, Na-EDTA, omega-3 fatty acid, and at least two proteaseinhibitors, thereby treating diabetes mellitus. In another embodiment,the present invention provides a method for treating diabetes mellitusin a human subject, comprising administering orally to the subject apharmaceutical composition comprising a Exenatide, Na-EDTA, omega-3fatty acid, and at least two protease inhibitors, thereby treatingdiabetes mellitus.

In one embodiment, the diabetes mellitus is Type I diabetes. In anotherembodiment, the diabetes mellitus is Type II diabetes. In anotherembodiment, the diabetes mellitus is insulin-dependent diabetes. Inanother embodiment, the diabetes mellitus is non-insulin-dependentdiabetes. In another embodiment, the diabetes mellitus is any other typeof diabetes known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, three treatments a day of the insulin composition areadministered. In another embodiment, two treatments a day areadministered. In another embodiment, four treatments a day areadministered. In another embodiment, one treatment a day isadministered. In another embodiment, more than four treatments a day areadministered. Each possibility represents a separate embodiment of thepresent invention.

Any of the methods of the present invention may utilize, in variousembodiments, any of the compositions of the present invention.

In another embodiment, the present invention provides a composition fororal administration of insulin, comprising an insulin protein and atleast two protease inhibitors, whereby a substantial fraction of theinsulin retains the enzymatic activity after absorption through anintestinal mucosal barrier of a human subject. In another embodiment,the present invention provides a composition for oral administration ofExenatide, comprising an insulin protein and at least two proteaseinhibitors, whereby a substantial fraction of the Exenatide retains theenzymatic activity after absorption through an intestinal mucosal batherof a human subject. In one embodiment, the present invention provides acomposition for oral administration of a protein, comprising a proteinand at least two protease inhibitors, whereby a substantial fraction ofthe protein retains the enzymatic activity after absorption through anintestinal mucosal barrier of the subject.

In one embodiment, the present invention provides the use of a proteinand at least two protease inhibitors in the manufacture of a medicamentfor oral administration of a protein with an enzymatic activity to asubject, whereby a substantial fraction of the protein retains theenzymatic activity after absorption through an intestinal mucosalbarrier of the subject. In one embodiment, the present inventionprovides the use of a protein, at least two protease inhibitors, and anomega-3 fatty acid in the manufacture of a medicament for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal bather of thesubject. In one embodiment, the present invention provides the use of aprotein, at least two protease inhibitors, Na-EDTA, and an omega-3 fattyacid in the manufacture of a medicament for oral administration of aprotein with an enzymatic activity to a subject, whereby a substantialfraction of the protein retains the enzymatic activity after absorptionthrough an intestinal mucosal bather of the subject.

In one embodiment, the present invention provides the use of an insulinprotein and at least two protease inhibitors in the manufacture of amedicament for treating diabetes mellitus in a subject. In oneembodiment, the present invention provides the use of an insulinprotein, at least two protease inhibitors, and an omega-3 fatty acid inthe manufacture of a medicament for treating diabetes mellitus in asubject. In one embodiment, the present invention provides the use of aninsulin protein, at least two protease inhibitors, Na-EDTA, and anomega-3 fatty acid in the manufacture of a medicament for treatingdiabetes mellitus in a subject.

In one embodiment, the present invention provides the use of a Exenatideprotein and at least two protease inhibitors in the manufacture of amedicament for treating diabetes mellitus in a subject. In oneembodiment, the present invention provides the use of a Exenatideprotein, at least two protease inhibitors, and an omega-3 fatty acid inthe manufacture of a medicament for treating diabetes mellitus in asubject. In one embodiment, the present invention provides the use of aExenatide protein, at least two protease inhibitors, Na-EDTA, and anomega-3 fatty acid in the manufacture of a medicament for treatingdiabetes mellitus in a subject.

In one embodiment, methods and compositions of the present inventionhave the advantage of more closely mimicking physiological insulinsecretion by the pancreas. When insulin is secreted into the portalvein, the liver is exposed to a greater insulin concentration thanperipheral tissues. Similarly, insulin administered according to thepresent invention reaches the intestine and is absorbed in the bodythrough the intestine and through the portal system to the liver. Thisabsorption route thus resembles the physiological secretion of insulinby the pancreas, enabling, in this embodiment, delicate control of theblood glucose level and the metabolic activities of the liver and theperipheral organs controlled by insulin. By contrast, when insulin isadministered to insulin-deficient diabetic patients via the peripheralvenous system, the concentration of insulin in the portal vein issimilar to that in the peripheral circulation, resulting inhypoinsulinemia in the portal vein and the liver and hyperinsulinemia inthe peripheral venous system. This leads, in one embodiment, to anabnormal pattern of glucose disposal.

In another embodiment, different constituents of compositions of thepresent composition are absorbed at different rates from the intestinallumen into the blood stream. The absorption of the bile acid, in oneembodiment, is significantly faster than the absorption of insulin.

For this reason, in another embodiment, a drug regimen involvingingestion of a pair of pills at spaced intervals, e.g., a second pillcontaining a higher concentration of enhancer is taken at a definedinterval (e.g. 30 minutes) after the first pill. In another embodiment,certain of the constituents are microencapsulated to enhance theabsorption of the insulin into the system. In another embodiment,certain of the constituents are microencapsulated to enhance theabsorption of the Exenatide into the system.

In one embodiment, a treatment protocol of the present invention istherapeutic. In another embodiment, the protocol is prophylactic. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, solid carriers/diluents for use in methods andcompositions of the present invention include, but are not limited to, agum, a starch (e.g. corn starch, pregeletanized starch), a sugar (e.g.,lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g.microcrystalline cellulose), an acrylate (e.g. polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

In another embodiment, the compositions further comprise binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g. cornstarch, potato starch, alginic acid,silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodiumstarch glycolate), buffers (e.g., Tris-HCL, acetate, phosphate) ofvarious pH and ionic strength, additives such as albumin or gelatin toprevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Plutonic F68, bile acid salts), protease inhibitors, surfactants (e.g.sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing agents (e.g. carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g. aspartame, citric acid),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g. stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g. colloidal silicon dioxide),plasticizers (e.g. diethyl phthalate, triethylcitrate), emulsifiers(e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymercoatings (e.g., poloxamers or poloxamines), coating and film formingagents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/oradjuvants. Each of the above excipients represents a separate embodimentof the present invention.

In some embodiments, the dosage forms of the present invention areformulated to achieve an immediate release profile, an extended releaseprofile, or a delayed release profile. In some embodiments, the releaseprofile of the composition is determined by using specific excipientsthat serve for example as binders, disintegrants, fillers, or coatingmaterials. In one embodiment, the composition will be formulated toachieve a particular release profile as known to one skilled in the art.

In one embodiment, the composition is formulated as an oral dosage form.In one embodiment, the composition is a solid oral dosage formcomprising tablets, chewable tablets, or capsules. In one embodiment thecapsules are soft gelatin capsules. In another embodiment, capsules asdescribed herein are hard-shelled capsules. In another embodiment,capsules as described herein are soft-shelled capsules. In anotherembodiment, capsules as described herein are made from gelatine. Inanother embodiment, capsules as described herein are made fromplant-based gelling substances like carrageenans and modified forms ofstarch and cellulose.

In other embodiments, controlled- or sustained-release coatings utilizedin methods and compositions of the present invention include formulationin lipophilic depots (e.g. fatty acids, waxes, oils).

The compositions also include, in another embodiment, incorporation ofthe active material into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts.) Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance. In another embodiment,particulate compositions of the active ingredients are coated withpolymers (e.g. poloxamers or poloxamines)

In another embodiment, the compositions containing the insulin andomega-3 fatty acid are delivered in a vesicle, e.g. a liposome (seeLanger, Science 249:1527-1533 (1990); Treat et al., in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see generally ibid). In another embodiment, the compositionscontaining the Exenatide and omega-3 fatty acid are delivered in avesicle, e.g. a liposome (see Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of infectious Disease andCancer, Lopez-Berestein and Fidler (ads.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

The preparation of pharmaceutical compositions that contain an activecomponent, for example by mixing, granulating, or tablet-formingprocesses, is well understood in the art. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active ingredients of compositions of the presentinvention are mixed with additives customary for this purpose, such asvehicles, stabilizers, or inert diluents, and converted by customarymethods into suitable forms for administration, such as tablets, coatedtablets, hard or soft gelatin capsules, aqueous, alcoholic or oilysolutions.

Each of the above additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

In one embodiment, the term “treating” refers to curing a disease. Inanother embodiment, “treating” refers to preventing a disease. Inanother embodiment, “treating” refers to reducing the incidence of adisease. In another embodiment, “treating” refers to amelioratingsymptoms of a disease. In another embodiment, “treating” refers toinducing remission. In another embodiment, “treating” refers to slowingthe progression of a disease.

EXPERIMENTAL DETAILS SECTION Example 1: Capsules Comprising a Proteinand a Combination of Protease Inhibitors Materials and ExperimentalMethods

Formulation

4 days prior to dosing, formulations were prepared containing: (1) 8 mginsulin, 150 mg EDTA, 125 mg SBTI in 1 ml fish oil in a soft-gel capsule(SwissCup), (2). 8 mg insulin, 150 mg EDTA, 150000 KIU Aprotinin in 1 mlfish oil in a soft-gel capsule (SwissCup), (3) 8 mg insulin, 150 mgEDTA, 150000 KIU Aprotinin, 125 mg SBTI in 1 ml fish oil in a soft-gelcapsule (SwissCup). The formulations were stored in the refrigerator (4°C.) until dosing.

Results

In the next experiment, the formulations as describes in the“Formulation” section were orally consumed by healthy human subjects. Asshown in FIG. 1 (A-C), blood glucose levels were significantly reducedand more stable in human subjects treated with formulation (3) (8 mginsulin, 150 mg EDTA, 150000 KIU Aprotinin, 125 mg SBTI in 1 ml fish oilin a soft-gel capsule (SwissCup). These results also suggest that thetwo protease inhibitors in formulation (3) had a synergistic effect inlowering blood glucose levels.

It should be emphasized that no differences in the reduction of bloodglucose levels were observed in a previous experiment in dogs treatedwith the same formulations comprising a single protease inhibitor(formulations 1 or 2, SBTI or Aprotinin) or a formulation that comprisesboth SBTI and Aprotinin (formulation 3). Thus, the results regarding thereduction in blood glucose levels with insulin and a combination ofprotease inhibitors was unexpected.

Furthermore, as shown in FIG. 1 D-F, total blood insulin wassignificantly higher especially between 220-300 minutes in humansubjects treated with formulation (3) (8 mg insulin, 150 mg EDTA, 150000KIU Aprotinin, 125 mg SBTI in 1 ml fish oil in a soft-gel capsule(SwissCup). These results also suggest that the two protease inhibitorsin formulation (3) had a synergistic effect in stabling insulin levels.

As shown in FIG. 1 G-I, blood C-peptide levels were significantlyreduced in human subjects treated with formulation (3) (8 mg insulin,150 mg EDTA, 150000 KIU Aprotinin, 125 mg SBTI in 1 ml fish oil in asoft-gel capsule (SwissCup). These results also suggest that the twoprotease inhibitors in formulation (3) had a synergistic effect inlowering blood C-peptide levels.

In a similar experiment in healthy humans, formulations comprising: (1)150 mg EDTA, 24 mg Aprotinin, 75 mg BBI, and 10 mg insulin; (2) 150 mgEDTA, 24 mg Aprotinin, 75 mg Kunitz, and 12 mg insulin; and (3) 150 mgof EDTA, 24 mg Aprotinin, 75 mg SBTI, and 8 mg of insulin were found tobe effective in lowering blood glucose levels and maintaining sufficientblood insulin levels for over 3 hours after a meal.

It should be emphasized that no differences in the reduction ofC-peptide levels were observed in a previous experiment in dogs treatedwith the same formulations comprising a single protease inhibitor(formulations 1 or 2, SBTI or Aprotinin) or a formulation that comprisesboth SBTI and Aprotinin (formulation 3). Thus, the results regarding thereduction in blood C-peptide levels with insulin and a combination ofprotease inhibitors was unexpected.

The above described formulations are also active with 0.5 ml fish oilboth in dogs and humans. Dogs do not react to Aprotinin. Therefore indogs the reduction in blood glucose with SBTI alone was equivalent tothe reduction in blood glucose with both Aprotinin and SBTI. Moreover, aformulation comprising insulin and Aprotinin but not SBTI did not reduceblood glucose in dogs.

Example 2: Optimization of Source of Omega-3 Fatty Acids

Various omega-3 fatty acids or sources of omega-3 fatty acids (e.g.those listed above in the specification) are compared for their abilityto preserve insulin following oral administration in methods andcompositions of the present invention. Insulin tablets or capsules areformulated as described in the above Examples, except that the insulinis dissolved in the alternate source instead of in fish oil. The mosteffective source of omega-3 fatty acids is used in subsequent Examples.

Example 3: Optimization of Protease Inhibitors

Various protease inhibitors (either non-toxic or having an acceptabletoxicity profile; e.g. those listed above in the specification) arecompared for their ability to preserve insulin following oraladministration in methods and compositions of the present invention.Insulin and/or Exenatide tablets or capsules are formulated as describedin the above Examples, except that the alternate protease inhibitors aresubstituted for SBTI and/or Aprotinin. Amounts of the proteaseinhibitors are also varied, to determine the optimal amounts. The mosteffective protease inhibitor/amount is used in subsequent Examples.

Example 4: Optimization of Enhancer

Various enhancers (e.g. those listed above in the specification) arecompared for their ability to facilitate absorption of insulin followingoral administration in methods and compositions of the presentinvention. Insulin tablets or capsules are formulated as described inthe above Examples, except that the alternate enhancers are substitutedfor EDTA. Amounts of the enhancers are also varied, to determine theoptimal amounts. The most effective enhancer/amount is used insubsequent experiments.

Example 5: Optimization of Type and Amount of Insulin

Various types and amounts of insulin e.g. those listed above in thespecification) are compared for their ability to regulate blood sugar inmethods and compositions of the present invention. Insulin tablets orcapsules are formulated as described in the above Examples, except thatthe type and amount of insulin is varied. The most effective type/amountof insulin is used in clinical trials.

What is claimed is:
 1. A method for increasing the bioavailability of aprotein having a molecular weight up to 100,000 Daltons in a subject,comprising orally administering, to the subject, a formulation thatcomprises a pharmaceutical composition, wherein the pharmaceuticalcomposition comprises: (i) the protein, wherein the protein issomatotropin, insulin growth factor-I, an oxidoreductase, a transferase,a hydrolase, a lyase, an isomerase, a ligase, an annexin, an ATP-bindingcassette transporter, hemoglobin, an ATPase, a calcium channel, apotassium channel, a sodium channel, a solute carrier, an albumin,lactoglobulin, casein, ovomucin, ferritin, phosvitin, lactoferrin,vitellogenin, amyloid, collagen, elastin, or fibrillin; (ii) at leasttwo protease inhibitors, wherein the protease inhibitors comprise atleast both soybean trypsin inhibitor (SBTI) and aprotinin; (iii) omega-3fatty acid; (iv) a substance that enhances absorption of the proteinthrough the subject's intestinal mucosal barrier, wherein the substanceis (a) ethylenediaminetetraacetic acid (EDTA) or a salt thereof, or (b)a bile acid or alkali metal salt thereof; and (v) an enteric or gelatincoating; and wherein the protease inhibitors increase thebioavailability of the protein when the composition is administeredorally to the subject.